Tks5/Fish is a scaffolding protein with five SH3 domains and one PX domain. In Src-transformed cells, Tks5/Fish localizes to podosomes, discrete protrusions of the ventral membrane. We generated Src-transformed cells with reduced Tks5/Fish levels. They no longer formed podosomes, did not degrade gelatin, and were poorly invasive. We detected Tks5/Fish expression in podosomes in invasive cancer cells, as well as in human breast cancer and melanoma samples. Tks5/Fish expression was also required for protease-driven matrigel invasion in human cancer cells. Finally, coexpression of Tks5/Fish and Src in epithelial cells resulted in the appearance of podosomes. Thus, Tks5/Fish appears to be required for podosome formation, for degradation of the extracellular matrix, and for invasion of some cancer cells.

Ferroportin and hepcidin are critical proteins for the regulation of systemic iron homeostasis. Ferroportin is the only known mechanism for export of intracellular non–heme-associated iron; its stability is regulated by the hormone hepcidin. Although ferroportin profoundly affects concentrations of intracellular iron in tissues important for systemic iron absorption and trafficking, ferroportin concentrations in breast cancer and their influence on growth and prognosis have not been examined. We demonstrate here that both ferroportin and hepcidin are expressed in cultured human breast epithelial cells and that hepcidin regulates ferroportin in these cells. Further, ferroportin protein is substantially reduced in breast cancer cells compared to nonmalignant breast epithelial cells; ferroportin protein abundance correlates with metabolically available iron. Ferroportin protein is also present in normal human mammary tissue and markedly decreased in breast cancer tissue, with the highest degree of anaplasia associated with lowest ferroportin expression. Transfection of breast cancer cells with ferroportin significantly reduces their growth after orthotopic implantation in the mouse mammary fat pad. Gene expression profiles in breast cancers from >800 women reveal that decreased ferroportin gene expression is associated with a significant reduction in metastasis-free and disease-specific survival that is independent of other breast cancer risk factors. High ferroportin and low hepcidin gene expression identifies an extremely favorable cohort of breast cancer patients who have a 10-year survival of >90%. Ferroportin is a pivotal protein in breast biology and a strong and independent predictor of prognosis in breast cancer.

Ovarian cancer is a lethal malignancy that has not seen a major therapeutic advance in over 30 years. We demonstrate that ovarian cancer exhibits a targetable alteration in iron metabolism. Ferroportin (FPN), the iron efflux pump, is decreased, and transferrin receptor (TFR1), the iron importer, is increased in tumor tissue from patients with high grade but not low grade serous ovarian cancer. A similar profile of decreased FPN and increased TFR1 is observed in a genetic model of ovarian cancer tumor initiating cells (TICs). The net result of these changes is an accumulation of excess intracellular iron and an augmented dependence on iron for proliferation. A forced reduction in intracellular iron reduces the proliferation of ovarian cancer TICs in vitro, and inhibits both tumor growth and intraperitoneal dissemination of tumor cells in vivo. Mechanistic studies demonstrate that iron increases metastatic spread by facilitating invasion through expression of matrix metalloproteases and synthesis of IL6. We show that the iron dependence of ovarian cancer tumor initiating cells renders them exquisitely sensitive in vivo to agents that induce iron-dependent cell death (ferroptosis) as well as iron chelators, and thus creates a metabolic vulnerability that can be exploited therapeutically.

Activation of ferroptosis, a recently described mechanism of regulated cell death, dramatically inhibits growth of ovarian cancer cells. Given the importance of lipid metabolism in ferroptosis and the key role of lipids in ovarian cancer, we examined the contribution to ferroptosis of steroyl CoA desaturase (SCD1), an enzyme that catalyzes the rate-limiting step in monounsaturated fatty acid synthesis, in ovarian cancer cells. SCD1 was highly expressed in ovarian cancer tissue, cell lines, and a genetic model of ovarian cancer stem cells. Inhibition of SCD1 induced lipid oxidation and cell death. Conversely, over-expression of SCD1 or exogenous administration of its C16:1 and C18:1 products, palmitoleic acid or oleate, protected cells from death. Inhibition of SCD1 induced both ferroptosis and apoptosis: inhibition of SCD1 decreased CoQ 10 , an endogenous membrane antioxidant whose depletion has been linked to ferroptosis, while concomitantly decreasing unsaturated fatty acyl chains in membrane phospholipids and increasing long chain saturated ceramides, changes previously linked to apoptosis. Simultaneous triggering of two death pathways suggests SCD1 inhibition may be an effective component of anti-tumor therapy, since overcoming this dual mechanism of cell death may present a significant barrier to the emergence of drug resistance. Supporting this concept, we observed that inhibition of SCD1 significantly potentiated the anti-tumor effect of ferroptosis inducers in both ovarian cancer cell lines and a mouse orthotopic xenograft model. Our results suggest that the use of combined treatment with SCD1

Ferroportin and hepcidin are critical proteins for the regulation of systemic iron homeostasis. Ferroportin is the only known mechanism for export of intracellular non-heme-associated iron; its stability is regulated by the hormone hepcidin. Although ferroportin profoundly affects concentrations of intracellular iron in tissues important for systemic iron absorption and trafficking, ferroportin concentrations in breast cancer and their influence on growth and prognosis have not been examined. We demonstrate here that both ferroportin and hepcidin are expressed in cultured human breast epithelial cells and that hepcidin regulates ferroportin in these cells. Further, ferroportin protein is substantially reduced in breast cancer cells compared to nonmalignant breast epithelial cells; ferroportin protein abundance correlates with metabolically available iron. Ferroportin protein is also present in normal human mammary tissue and markedly decreased in breast cancer tissue, with the highest degree of anaplasia associated with lowest ferroportin expression. Transfection of breast cancer cells with ferroportin significantly reduces their growth after orthotopic implantation in the mouse mammary fat pad. Gene expression profiles in breast cancers from >800 women reveal that decreased ferroportin gene expression is associated with a significant reduction in metastasis-free and disease-specific survival that is independent of other breast cancer risk factors. High ferroportin and low hepcidin gene expression identifies an extremely favorable cohort of breast cancer patients who have a 10-year survival of